Controlled sequential in situ self-assembly and disassembly of a fluorogenic cisplatin prodrug for cancer theranostics

Temporal control of delivery and release of drugs in tumors are important in improving therapeutic outcomes to patients. Here, we report a sequential stimuli-triggered in situ self-assembly and disassembly strategy to direct delivery and release of theranostic drugs in vivo. Using cisplatin as a model anticancer drug, we design a stimuli-responsive small-molecule cisplatin prodrug (P-CyPt), which undergoes extracellular alkaline phosphatase-triggered in situ self-assembly and succeeding intracellular glutathione-triggered disassembly process, allowing to enhance accumulation and elicit burst release of cisplatin in tumor cells. Compared with cisplatin, P-CyPt greatly improves antitumor efficacy while mitigates off-target toxicity in mice with subcutaneous HeLa tumors and orthotopic HepG2 liver tumors after systemic administration. Moreover, P-CyPt also produces activated near-infrared fluorescence (at 710 nm) and dual photoacoustic imaging signals (at 700 and 750 nm), permitting high sensitivity and spatial-resolution delineation of tumor foci and real-time monitoring of drug delivery and release in vivo. This strategy leverages the advantages offered by in situ self-assembly with those of intracellular disassembly, which may act as a general platform for the design of prodrugs capable of improving drug delivery for cancer theranostics.

Supplementary Figure 12. Immunofluorescence staining of ALP expressed on the membranes of HeLa cells. HeLa cells were first stained with the primary antibody of ALP (tissue nonspecific ALP (TNAP)), and then stained with the Alexa Fluor ® 488labeled secondary antibody (goat anti-rabbit IgG (H+L)). Nuclei were then stained with Hoechst 33342. The confocal fluorescence images were then acquired at Alexa Fluor ® 488 (green) and Hoechst 33342 (blue) channels, respectively. White arrows indicate the expression of membrane-bound TNAP on HeLa cells. Scale bar: 25 μm.
One representative experiment out of three is shown.  Figure 13. NIR FL imaging of HeLa cells upon incubation with P-CyPt (10 μM) for 5-60 min. HeLa cells were incubated with P-CyPt (10 μM) for 5-60 min, and co-stained with Hoechst 33342 (blue). After being washed, the FL images were acquired under NIR channel (λex/em = 670/750 ± 50 nm) and Hoechst channel (λex/em = 405/450 ± 20 nm), respectively. NIR FL (red) progressively appeared around cell membrane, and reached the maximum after 30 min. Some punctuate FL appeared intracellularly after 40 min. Scale bars: 25 μm. One representative experiment out of two is shown. Figure 14. NIR FL imaging of HeLa cells following incubation with varying concentration of P-CyPt for 30 min. HeLa cells were incubated with P-CyPt (0.1, 0.5, 5.0, 10.0 μM) for 30 min, and co-stained with Hoechst 33342 (blue). After being washed, the FL images were acquired at NIR channel (λex/em = 670/750 ± 50 nm) and Hoechst channel (λex/em = 405/450 ± 20 nm), respectively. NIR FL (red) increased with the concentration of P-CyPt. Scale bars: 25 μm. One representative experiment out of two is shown. Figure 15. Co-localization study of P-CyPt in HeLa cells. HeLa cells were incubated with P-CyPt (10 μM, red) for 1 h, and then stained with the Lysotracker (cyan) and Hoechst 33342 (blue). The imaging results showed that the punctate intracellular NIR fluorescence (red) colocalized well with the fluorescence of lysotracker, indicating the main distribution of dephosphorylated fluorescent product in the lysosomes in addition to the cell membranes. Scale bars: 10 μm. One representative experiment out of two is shown. Figure 16. Examination of endocytosis pathway in HeLa cells. NIR fluorescence imaging of HeLa cells upon pretreatment with different endocytosis inhibitor, following by incubation with P-CyPt (10 μM) for 60 min. To inhibit endocytosis, HeLa cells were first incubated with chlorpromazine (CPZ, 50 μM), ethylisopropyl amiloride (EIPA, 100 μM), or filipin III (5 μg/mL) for 30 min, and then incubated with P-CyPt (10 μM) for 60 min. White arrows indicate the appearance of fluorescent punctuates in the interior of HeLa cells. HeLa cells pretreated with CPZ showed reduced intracellular fluorescence, which was not largely inhibited by EIPA or filipin III. These imaging results indicate that the in situ formed Pt IV NPs can enter HeLa cells mainly via the clathrin-dependent endocytosis, not via micropinocytosis (EIPA) or caveolae-mediated endocytosis (filipin III). Scale bars: 25 μm. One representative experiment out of two is shown. Figure 17. Evaluation of the different ability to anchor on cell membranes between in situ formed Pt IV NPs and preformed Pt IV NPs. Confocal fluorescence imaging of HeLa cells following incubation with P-CyPt (10 μM) or preformed Pt IV NPs (10 μM) for 30 min. (a) The images were acquired before wash (Pre-wash). (b) The images were acquired after removal of medium and washed gently with PBS buffer (Post-wash). Scale bars: 25 μm. These results suggest that the ALPmediated in situ self-assembled Pt IV NPs are prone to adhere on the membranes of HeLa cells, while preformed Pt IV NPs could not adhere on the cell membranes. One representative experiment out of two (a,b) is shown. Figure 18. NIR FL/PA bimodality imaging of different culture mediums collected from indicated cells. (a) Photograph, FL images, and dual PA images (700&750 nm), (b) normalized NIR FL intensity, (c) normalized dual PA intensities, and (d) HPLC traces (660 nm detection) of different culture mediums. I: HeLa cells incubated with P-CyPt (10 μM) for 30 min; II: HeLa cells incubated with P-CyPt (10 μM) for 30 min, then washed with PBS three times, added blank medium and incubated for another 3 h; III: HeLa cells pretreated with Na3VO4 (10 mM) for 20 min, followed by incubating with P-CyPt (10 μM) for another 30 min; IV: HeLa cells incubated with preformed Pt IV NPs (10 μM) for 30 min; V: HEK-293T cells were incubated with P-CyPt (10 μM) for 30 min. Data denote mean ± s.d. (n = 3 independent cell pellets).

Supplementary Figure 19. Evaluation of time-dependent intracellular translocation of in situ formed Pt IV NPs in HeLa cells.
Confocal fluorescence images of HeLa cells incubated with P-CyPt (10 μM) for 30 min, and then washed with PBS for three times, followed by incubation with blank DMEM medium for 1, 3, 7.5 and 23.5 h. Scale bars: 25 μm. The resutls show that NIR FL was first distributed around cell membrane, which was mostly translocated into cells after 3 h. The intracellular NIR fluorescence became much brighter after elongation to 23.5 h, presumably owing to the GSH-triggered disassembly of Pt IV NPs intracellularly. Moreover, bright field (BF) images also showed that a distinct change in cell morphology after 23.5 h. One representative experiment out of two is shown. (a) Agarose gel electrophoresis of supercoiled pBR322 plasmid DNA (10 ng/μL) after incubating with indicated conditions. 1kb-I represents the linear DNA marker ladder (500-12000 bp); Lane i: DNA; Lane ii: DNA + GSH (2 mM); Lane iii: DNA + CDDP (100 μM); Lane iv: DNA + CDDP (100 μM) + GSH (2 mM); Lane v: DNA + P-CyPt (100 μM); Lane vi: DNA + P-CyPt (100 μM) + GSH (2 mM), Lane vii: DNA +P-CyPt (100 μM) + ALP (500 U/L), Lane viii: DNA +P-CyPt (100 μM) + ALP (500 U/L) +GSH (2 mM); (b) Agarose gel electrophoresis of supercoiled pBR322 plasmid DNA (20 ng/μL) after incubating with varying concentrations of P-CyPt (0, 1, 2, 5, 10, 20, 50 and 100 μM) respectively) plus ALP (500 U/L), followed by incubation with GSH (2 mM) in Tris buffer at 37 ºC for 16 h. The results in (a) demonstrate that: 1) CDDP itself cannot obviously relax or break the supercoiled DNA (Form I), but can bind to the DNA double strand and retard DNA movement in the gel (iii); 2) the addition of GSH into CDDP can scavenge CDDP and prevent its binding with the DNA double strand (iv); 3) P-CyPt itself cannot bind to and break the DNA double strand (v), but the addition of GSH to reduce Pt(IV) in P-CyPt can significantly relax the supercoiled DNA (Form II: open circular DNA), and partially break the DNA double strand (Form III: linear DNA) (vi); 4) Pt IV NPs that are formed by ALP-triggered dephosphorylation and in situ self-assembly of P-CyPt cannot bind to and break the DNA double strand neither (vii); 5) the addition of GSH to reduce Pt(IV) in the as-formed Pt IV NPs can relax the supercoiled DNA (Form II) and partially break the DNA double strand (Form III) as well (viii). These data suggest that GSHtriggered reduction and release of Pt(II) can efficiently relax the supercoiled DNA and break the DNA double strand, which is highly dependent on the concentration of P-CyPt (b). One representative experiment out of two is shown. Figure 22. Schematic illustration of the mechanism affecting the different uptake and intracellular accumulation of Pt drugs between free cisplatin (a) and P-CyPt (b). As shown in (a), cisplatin is highly polar and enters cells relatively slowly compared to other classes of small-molecule drugs. Though the uptake route of cisplatin into cells is not completely understood, it was reported that cisplatin could enter cells mainly via passive diffusion, and some plasma-membrane transporters (e.g., copper transporter CTR1) mediated active uptake may also be involved (Nat. Rev. Cancer 2007, 7, 573;Coord. Chem. Rev. 2009, 253, 2070. Once it enters cells, it is mainly distributed in the cytosol, where it becomes activated via displacing the chloride atoms by water molecules. This hydrolyzed product is a potent electrophile that can react with intracellular nucleophiles, including the reactive sulfhydryl groups in reducing biothiols (e.g., GSH) and proteins in the cytosol. Thus, the cisplatin is mainly captured in the cytosol, and only a few of cisplatin (and its hydrolyzed product) can enter nucleus and react with the nitrogen donor atoms in DNA (Eur. J. Pharmacol. 2014, 740, 364). Different to cisplatin, cellular uptake of the in situ formed Pt IV NPs is mainly via the clathrin-dependent endocytosis process (b). Once they enter cells, Pt IV NPs first reach the lysosomes, and are then reduced by abundant endogenous GSH, leading to disassembly and burst release of cisplatin in the lysosomes. Some of the released cisplatin can escape into the cytosol. This process could dramatically increase the intracellular concentration of cisplatin, which might outcompete the nucleophiles (e.g., GSH, free sulfhydryl-containing proteins) in the cytosol. Meanwhile, as the intracellular GSH is also depleted accompanying by the disassembly and succeeding release of cisplatin, cisplatin that is trapped by GSH could be reduced in the cytosol.

Supplementary
These two effects can allow more cisplatin to diffuse into other organelles (e.g., nucleus and mitochondria). Thus, the distributions of Pt in nucleus, lysosomes and mitochondria are found to be higher in cells incubated with P-CyPt than cisplatin.  MCTS were incubated with Pt IV NPs (10 μM) for 1 h, then washed with PBS buffer for three times, followed by incubation for another 24 h. The MCTs were then co-stained with PI. The FL images at mCy (λex/em = 670/710 nm, red) and PI channel (λex/em = 550/640 nm, magenta) were acquired using a confocal laser scanning microscopy. Scale bars: 100 μm. One representative experiment out of two is shown. These imaging results demonstrate that preformed Pt IV NPs hold a reduced ability to penetrate and induce cell death in HeLa MCTS compared to that of the smallmolecule P-CyPt. Pt, 200 μL) or P-CyPt (2.25 mg kg -1 Pt, 200 μL) at day 0, 3, 6, ,9 and 12. Then, all mice were kept feeding for another 9 days. On day 21, the mice were sacrificed. The main organs, including heart, liver, kidneys, spleen and lung were resected, cut into 10-μm slices, and applied for H&E staining. Red arrows indicate the damage regions occurred in the liver of Pt IV NPs-treated mice and in the kidneys of CDDP-treated mice, respectively. In CDDP-treated group, we observed cast formation (red arrow) and widened Bowman's spaces (yellow arrows), implying the occurrence of nephrotoxicity in mice after treatment with CDDP. In Pt IV NPs-treated group mice, we observed decreased nucleus, dilation sinusoids (red arrows) and inflammatory cell infiltration (yellow arrow) in the liver tissue, suggesting the occurrence of hepatotoxicity in mice following continuous treatment with Pt IV NPs. In contrast, all organs looked normal between PBS-and P-CyPt-treated mice. These data demonstrate that P-CyPt holds low side-toxicity compared with CDDP or Pt IV NPs. Scale bar: 100 μm. The occurrence of hepatotoxicity in Pt IV NPs-treated mice could be presumably owing to (1) the large size of Pt IV NPs (~160 nm) that caused a high liver uptake and (2) the fast release of cytotoxic cisplatin from the liver-resided Pt IV NPs by hepatic GSH that led to a high concentration of cisplatin in the liver. One representative experiment out of two is shown.   1.84 ± 0.68 a Preformed Pt Ⅳ NPs (2.25 mg kg -1 Pt) were i.v. injected into mice, and the urine was collected at 0-2 h, 2-4 h, 4-8 h and 8-12 h, respectively. CyPt and its metabolites and its metabolites in the urine were quantified by analytical HPLC (detection at 660 nm). Percentages (%) of CyPt and Cy-COOH were calculated by dividing the amount (mol) of each compound in the urine to the amount (mol) of Pt Ⅳ NPs injected. Values denote mean ± s.d. (n = 3 independent experiments).

Supplementary
where , , , and denote the position, velocity, mass of the i particle, and the force acting on it, respectively. The total force acting on particle i is composed of conservative force F C , random force F R , and dissipative force F D , each of them is pairwise interactions. The total force is given by The sum of force acts over all particles within a certain cutoff radius rC, beyond which the force is neglected. Here rC is the only length-scale in the system, which is considered as the unit of length, rC = 1. The conservation force of two particles is soft-repulsive interaction acting along the line of the centers of two particles: where aij is the interaction parameter between particles i and j. The r-dependent weight function ω(rij) provides the range of interaction for DPD particles with a commonly used choice: 1 / for and 0 for > . , , and ̑ / . The dissipative force which is proportional to the relative velocity, , is defined as where γ is the friction coefficient controlling the magnitude of the dissipative force. The random force acting as a heat source to equilibrate the thermal motion of unresolved scales is given by Where σ is the noise amplitude governing the intensity of the random force and θij(t) is a randomly fluctuating variable with Gaussian statistics: 0 and .
Español and Warren 1 showed that the two weight functions and can be chosen arbitrarily and this choice fixes the other weight function, with the relation as shown in the following equation: As a simple choice, we take , i.e. is the same function as in the conservative force. There is also a relation between the two amplitudes and kBT : 2 .
The combined effect of the dissipative and random force amounts to that of a thermostat.
The generic coarse-grained model ( Supplementary Fig. 3a) is constructed based on P-CyPt and CyPt. There are three different types of DPD particles, A, B and C for Pt group, Cy group and P group, respectively. For simplicity, all beads have the same size of rc and the same mass of m = 1. Within P-CyPt and CyPt, we use a finitely extensible nonlinear elastic (FENE) potential between the consecutive particles (Phys. Rev. Lett. 2000Lett. , 85, 1128Lett. -1131: VFENE(rij) = −1/2kR 0 2 ln[1−rij/ R0) 2 ] for rij < R0 and VFENE(rij) = ∞ for rij ≥ R0. In our simulation, we set k = 50 and R0 = 1.5rc. The time scale is set to τ = (mrc 2 / kBT) 1/2 , and the energy scale is given by kBT = 1, where kB is Boltzmann constant and T is the temperature. We use the modified Velocity-Verlet algorithm with λ = 0.65 in integrating the equation of motion. What's more, we set time step Δt= 0.03τ and the amplitude of random noise σ = 3.0 to avoid divergence of the simulation.
The molecules P-CyPt and CyPt can be represented by amphiphilic copolymer ABC and AB, which are dissolved in the solvent S, where S is the good solvent for A and C. The length of the block NB is fixed at 8. The interaction parameters chosen are shown in the following symmetric matrix: When we fix the three-dimension simulation with a fixed system number density of 3.0, the relationship between the aij and Flory-Huggins interaction parameters is: 3.497 .
We set aii = 25 for the same type of DPD particles (i = A, B, C, S), guaranteeing the correct excluded volume of the molecules. aij rises from 25 with increasing the incompatibility between particles i and j. Considering the relative position and chemical components of all the beads, we always keep aAB = aAC = 28, aAS = aCS = 25, and aBS = 35. The concentration of ABC or AB is fixed at 0.03. We performed the dynamics of total 81 000 DPD beads in a cubic box (30 3 ) under the periodic boundary conditions. In these conditions, we carry out 1.0 × 10 6 steps for each simulation.
Molecular simulation using the Materials Studio program. For each molecule, the most favourable crystal packing was predicted using the Polymorph module as implemented in Materials Studio. The crystal structure prediction protocol includes three parts, Packing, Geometry Optimization and Clustering. The main part of the procedure is the Packing phase, in which the space of possible crystal structures is searched for candidates of low energy. In addition, the space groups of interest must be provided (usually a small subset is required, not the full set of 230 space groups). In the present study, the most frequent 10 space groups (P21/c, P-1, P212121, C2/c, P21, Pbca, Pna21, Cc, Pbcn and C2) were provided in the Packing phase, and Monte Carlo Simulated Annealing was used as the search algorithm. Smart algorithm was used in the Geometry Optimization phase. Ewald summation method and Atom based summation method were used for electrostatic and van der Waals interactions, respectively. All energy calculations were performed with the Universal force field. LogP measurement in solutions. P-CyPt or CyPt (0.2 mM) in 1.0 mL octanol was subjected to partition with 1.0 mL octanol-saturated water. The resulting mixture was stirred vigorously for 5 min and centrifuged at 2040 × g for 5 min. Then, the octanol layer and water layer were separated. The UV-vis absorption spectroscopy or fluorescence spectrum was recorded after being diluted in MeOH/H2O (1:1) system (<1% octanol). The logP value was calculated by dividing the amount in the octanol layer to that in water layer.

Evaluation of the dephosphorylated efficacy of P-CyPt towards
Release of CDDP from Pt IV NPs during disassembly process. P-CyPt (10 μM) in 5 mL Tris buffer was incubated with ALP (100 U/L) at 37 °C for 30 min to form Pt IV NPs, followed by addition with 10 mM GSH at 37 ºC. After 10, 20, 30, 40, 50 and 60 min, 50 mM N-ethylmaleimide was added into the solution to scavenge GSH. The reaction solution was centrifuged at 13,000 × g and the supernatant was collected. The supernatants were digested with concentrated HNO3 (65~70%) and HCl (36~38%) at 90 °C for 24 h. The solution was diluted to 5 mL 2% HNO3 and 5% HCl solution, and the concentration of Pt element was measured by ICP-OES, which was further applied to calculate the amount of CDDP (denoted as NPt) released in the solution. The percentage of Pt release at each time point was calculated according to the formula: Pt release % = NPt/N0 × 100% (9) Where N0 represents the total amount of Pt in the the in situ formed Pt IV NPs, and NPt represents the amount of Pt released from Pt IV NPs following incubation with GSH.
Characterization of enzyme kinetics of P-CyPt towards ALP. Briefly, varying concentrations of P-CyPt (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 μM) in 100 μL Tris buffer were placed in a 96-well black plate. The reactions were initiated upon addition of 100 μL ALP (30 U/L) to each solution. The resulting fluorescence intensities in each well in the first five minutes were recorded on a Spark™ 10M Multimode Microplate Reader (λex/em = 680/710 nm). The amount of CyPt at each time point was determined by a standard curve under the same conditions. Kinetics values, including Km and Vmax, were determined according to the Lineweaver-Burk plot.

Determination of the sensitivity of P-CyPt towards ALP.
To evaluate the sensitivity of P-CyPt towards ALP, P-CyPt (10 μM) was incubated with varying concentrations of ALP (0, 0.5, 1, 2, 5, 10, 15, 20, 50, 100, 150 and 200 U/L) in Tris buffer at 37 °C for 30 min, and the fluorescence spectra were then recorded on a HORIBA Jobin Yvon Fluoromax-4 fluorometer, with an excitation at 680 nm. The resulting fluorescence intensities at 710 nm were plotted to the concentrations of ALP, and a linear regression fitted from 0 -20 U/L ALP was obtained, affording the slope k. The detection limit was calculated from 3σ/k, where σ represents the standard deviation of 11 blank measurements.
Determination of the specificity towards ALP. The specificity towards ALP in vitro was evaluated based on fluorescence, respectively. P-CyPt (10 μM) in Tris buffer was incubated with 10 nmol/L of MMP-2, 100 U/L of γ-glutamyl transferase (GGT), 100 U/L Cathepsin B (CTB), 0.2 μg/mL of Caspase-3/7, 2.5 mg/mL of Trypsin, 100 U/L of ALP or 100 U/L ALP pretreated with its inhibitors, Na3VO4 (100 μM) for 10 min. The solutions were kept at 37 °C for 30 min. The fluorescence spectra were then acquired with an excitation at 680 nm.
Immunofluorescence staining of membrane-bound ALP. Briefly, HeLa cells were grown onto at glass-bottom dish and allowed to grow overnight. The cells were fixed with 4% paraformaldehyde for 15 min, treated with 0.1% Triton X-100-PBS for 1 h, and then blocked with 1% bovine serum albumin (BSA)/10 % normal goat serum. Then, the fixed HeLa cells were incubated with the primary antibody of tissue nonspecific ALP (Mouse monoclonal [2F4] to ALP, Tissue Non-Specific, ab126820, lot: GR3428682-4, dilution 1:100) at 4°C overnight. After being washed with PBS three times, the cells were then incubated with the Alexa Fluor®488-labeled secondary antibody (Alexa Fluor®488 goat anti-rabbit IgG (H+L), ab150077, lot: GR3376391-4, dilution 1:1000) at room temperature for 1 h. After that, Hoechst 33342 was used to stain the cell nuclei (blue). Last, cells were washed with PBS three times and mounted for imaging. The confocal fluorescence using the IX73 optical microscope equipped with DAPI and FITC filters, microscope (Olympus, IX73) with U plan supper apochromat objective 60x/1.35 oil lens (Olympus), and captured at 8 bit on a DP80 detector (Olympus), the analysis of the pictures captured using the microscope equipped with DAPI and FITC filters.
General procedure for fluorescence imaging of cells. Cells (~ 5×10 4 ) were seeded onto a glass-bottom dish (In Vitro Scientific, D35-20-1-N) and allowed to grow overnight. P-CyPt, or Pt IV NPs (10 μM) in FBS free DMEM was added into dishes and incubated at 37 °C for 30 or 60 min. To inhibit ALP activity, cells were pretreated with ALP inhibitor Na3VO4 (10 mM) for 20 min, and then incubated with P-CyPt (10 μM) for another 30 or 60 min. The medium was removed, gently washed with 1 mL PBS buffer once. After adding fresh medium, Fluorescence images were captured on a Leica TCS SP8 confocal laser scanning microscope, microscope (TCS SP8 STED 3X) with 63x/1.4 oil lens (Leica), and captured at RGB on a HyD detector (Leica Dmi8), with the excitation wavelength at 670 nm, and the emission wavelength from 690 nm to 750 nm.

Co-localization study.
To examine the intracellular location, HeLa cells were incubated with 2 μM Hoechst 33342 and 200 nM Lyso Tracker Green DND-26 for 20 min. After being washed with PBS for three times, the cells were then incubated with 10 μM P-CyPt for another 1 h. The cells were washed gently with 1 mL PBS buffer once. After adding fresh DMEM medium, the fluorescence images of the cells were captured on the confocal laser scanning microscope, using blue, green and NIR channels.
Investigation of Endocytosis pathway. Three endocytosis inhibitors, including chlorpromazine (CPZ) for clathrin-mediated endocytosis, filipin III for caveolaemediated endocytosis and ethylisopropyl amiloride (EIPA) for micropinocytosis were used to determine the possible pathways of cellular uptake of assembled Pt IV NPs. HeLa cells were treated with respective inhibitor (30 μM CPZ, 5 μg/mL filipin III or 100 μM EIPA) for 30 min, washed with PBS for three times, and then treated with 10 μM P-CyPt for another 1 h. After being washed with 1 mL PBS once, the fluorescence images were acquired using the confocal laser scanning microscope.
Bimodality FL and PA imaging of cell pellets. To examine the ability for FL and PA bimodality imaging of ALP activity in cells, HeLa or HEK29T cells were seeded in 10cm dishes at a density of 4×10 6 cells/well and allowed to grow overnight. P-CyPt or Pt IV NPs (10 μM) in 4 mL FBS free DMEM was added into wells and incubated at 37 °C for 30 min. To inhibit ALP activity, cells were pretreated with inhibitor Na3VO4 (10 mM) for 20 min, and then incubated with P-CyPt (10 μM) for another 30 min. To evaluate the disassembly of in situ formed Pt IV NPs of P-CyPt in response of intracellular GSH, P-CyPt (10 μM) in 4 mL FBS free DMEM was added into wells and incubated at 37 °C for 30 min, then the medium was removed and the cells were incubated for another 3 h after adding fresh medium. Then, the medium was removed, and cells were washed gently with 1 mL PBS once. Trypsin (1 mL) was added into each dish, maintained at 37 °C for 2 min to detach the cells. The cell pellets were then collected after centrifugation at 161× g for 4 min. The fluorescence imaging and PA imaging of the cell pellets were then acquired on an IVIS Lumina XR III system (λex/em = 670/750 ± 50) and VisualSonics Vevo 2100 LAZR system at 700 and 750 nm, respectively.
HPLC analysis of cell pellets. HeLa or HEK29T cells were seeded in 6-cm dishes at a density of 1×10 6 cells/well and allowed to grow overnight. P-CyPt or Pt IV NPs (10 μM) in 2 mL FBS free DMEM was added into wells and incubated at 37 ºC for 30 min. To inhibit ALP activity, cells were pretreated with inhibitor Na3VO4 (10 mM) for 20 min, and then incubated with P-CyPt (10 μM) for another 30 min. To evaluate the disassembly of in situ formed Pt IV NPs of P-CyPt in response of intracellular GSH, P-CyPt (10 μM) in 4 mL FBS free DMEM was added into wells and incubated at 37 °C for 30 min, then the medium was removed and the cells were incubated for another 3 h after adding fresh medium. Then, the corresponding culture mediums were collected. The cells were trypsinized, centrifuged and counted. After being lysed with 200 μL DMSO, the cell lysates were mixed with 300 μL cold MeOH and 500 μL D.I. water, and centrifuged at 13,000× g at 4 °C for 10 min. Aliquots of supernatant containing cell lysates (250 μL) and 500 μL of culture medium were injected into an HPLC system for analysis, respectively.
To establish the standard curve, varying concentrations of P-CyPt, CyPt or Cy-COOH (0.5-10 μM) was injected into analytical HPLC, and the corresponding peak area at each concentration was integrated to give a linear standard curve of HPLC peak area versus the concentration of P-CyPt, CyPt or Cy-COOH. Using the established standard curves, the concentrations of P-CyPt, CyPt and Cy-COOH in the lysates could be calculated, which were further divided by the number of cells, giving the cellular uptake of them (fmol/cell).

TEM analysis of the in situ self-assembled Pt IV NPs in HeLa cells.
To further demonstrate the formation of NPs in culture HeLa cells, HeLa cells were seeded on a 6-well cell culture plats at a density of 2×10 5 cells/well. After growing overnight, the cells were incubated with or without P-CyPt (10 μM) for 1 h and washed gently with cold PBS buffer for three times. Afterwards, trypsin (500 μL/well) was added into each well, maintained at 37 °C for 2 min to detach the cells. The cell pellets were then collected, and lysed via repeated freezing and thawing in liquid nitrogen. The cell lysates were separated according to a reported fragmentation procedure to obtained different cell fractions: pellet sample N (nuclei) was separated by 600 g for 10 min; pellet sample L (lysosomes and mitochondria) separated by 15000 g for 5 min; pellet sample M (plasma membrane) separated 100000g for 60 min and the remaining supernatant was soluble portion cytoplasm named as cell pellet C (cytosol) Pellets N, L, and M were all re-dispersed in 200 μL of D.I. water. Then, the solutions were collected and dropped on a carbon-coated copper grid, followed by freeze-drying respectively. The samples were then examined in a JEM-2800 Transmission Electron Microscope.

Quantitative analysis of Pt in HeLa cells and different subcellular organelles.
HeLa cells were seeded in 10-cm dishes at a density of 4×10 6 cells/well and allowed to grow overnight. The cells were incubated with P-CyPt (10 μM), Pt IV NPs (10 μM) and CDDP (10 μM) for 1 h, respectively. In P-CyPt + Na3VO4 group, HeLa cells were first incubated with Na3VO4 (10 mM) for 20 min, and then incubated with P-CyPt (10 μM) for another 60 min. After being washed with PBS for three times, the cells were trypsinized, centrifuged and counted. For Pt distribution analysis, the cells were treated with P-CyPt (10 μM) or CDDP (10 μM) for 1 h, and then washed with PBS for three times, followed by incubation with fresh medium for another 24 h. After that, cells were washed gently with 2 mL PBS once. The cells were trypsinized, centrifuged and counted. The cell lysates were separated according to a reported fragmentation procedure to obtained different cell fractions, including N (nuclei), M (plasma membrane), L (lysosomes and mitochondria) and C (cytosol). The Cells and different cell fractions were digested with concentrated HNO3 (65~70%) and HCl (36~38%) at 90 °C for 24 h. The solution was diluted to 5 mL 2% HNO3 and 5% HCl solution, and the concentration of Pt element was measured by ICP-OES, which was further applied to calculate the amount of Pt in cells and subcellular organelles.
Flow cytometry analysis of HeLa cell death. Approximately 2×10 5 cells/well HeLa cells in 2 mL DMEM medium were seeded onto 6-well plates and allowed to grow overnight. The medium was replaced with 1 mL of fresh medium containing P-CyPt (20 μM), Pt IV NPs (20 μM) and CDDP (20 μM), respectively. After 2 h incubation, medium was removed and cells were washed with PBS for three times. Then, cells were incubated with fresh medium for another 48 h to induce different levels of apoptosis. After gentle washing with PBS (×1), the cells were detached from the plates with trypsin (500 μL per well), and the cell pellets were collected after centrifugation at 1000 161× g at 4 °C for 4 min. After washing with PBS, the pellets were re-suspended in 500 μL PBS buffer and stained with the annexin V-FITC/PI double staining apoptosis detection kit for flow cytometry. All assays were performed according to the manufacturer instructions, and the cell population was analyzed by annexin V-FITC and PI channels. The count number for each flow cytometry analysis was ~5000 to 10000, and the data was processed using FlowJo software.

Depletion of intracellular GSH.
HeLa cells (2×10 5 cells/well) in 2 mL DMEM medium were seeded onto 6-well plates and allowed to grow overnight. Then cells were then treated with P-CyPt, Pt IV NPs, or CDDP (10 μM), respectively. After that, cells were digested with trypsin and washed with PBS for 3 times. Then, cell pellets were dispersed in pre-chilled extraction solution (1 mL, 0.1 M potassium phosphate buffer containing 0.1% Triton, 0.6% sulfosalicylic acid, and 5 mM EDTA, pH 7.4) and sonicated with an US probe on ice for 2 min. To ensure that cells were lysed completely, the solution was further frozen and thawed twice. Finally, the solution was centrifuged at 3000 g at 4 °C for 4 min. The supernatant was taken to measure the GSH content using commercial GSH detection kit, according to a reported procedure (Nat. Protoc.  2006, 1, 3159).
MCTS imaging and cytotoxicity. The three-dimensional multicellular spheroids (MCTS) of HeLa were cultured according to the method previously described (Nat. Protoc. 2009, 4, 309) with minor modifications. Briefly, a well of 96-well plates was covered by 50 μL of hot agarose solution (1.5 w/v %) and then cooled to form a layer of agaropectin. HeLa cells were seeded to the agaropectin-coated well at a density of 2×10 3 cells/well, and incubated to grow into spheroids. DMEM medium was replaced with fresh medium every 2 days until the diameter of tumor spheroids grows near 400 μm. Then, the MCSs were incubated with 10 μM P-CyPt or Pt IV NPs for 1 h, washed with PBS for 3 times. Blank DMEM medium was added, and incubated for another 24 h. After that, PI (4 μM) was added and incubated for 1 h. The medium was removed, and the spheroids were gently washed with PBS (×1), which was then carefully transferred to a glass bottom dish. The fluorescence images of the MCTS were captured on a Leica TCS SP8 confocal laser scanning microscope, microscope (TCS SP8 STED 3X) with 10x/0.4 dry lens (Leica), and captured at RGB on a HyD detector (Leica Dmi8) under PI and Cy5.5 channels.
Ex vivo FL imaging of resected organs. Mice bearing s.c. HeLa tumors were i.v. injected with P-CyPt (100 μM) or Pt IV NPs (100 μM) in 200 μL saline, and the mice were sacrificed after 4 h. Tumors and main organs, including liver, kidneys, intestines, heart, lung, stomach and spleen were resected. The fluorescence images of these organs and tumors were acquired with the IVIS Lumina XR III imaging system using a 660 nm excitation filter and a 750 ± 50 nm emission filter. Each experiment was conducted in three mice.
Biodistribution study of Pt. HeLa tumor-bearing mice were i.v. injected with P-CyPt (100 μM), Pt IV NPs (100 μM) or CDDP (100 μM) in 200 μL saline. Mice were sacrificed at 4 h post injection. Tumors and major organs including heart, liver, spleen, lung, and kidneys, were collected and weighed. The tissues were cut into small pieces and digested with concentrated HNO3 (65-70%, 2 mL) and HCl (36~38%, 1 mL) at 90 °C overnight. The residue in each organ was then diluted with 5 mL 2% HNO3 and 5% HCl solution, and the concentration of Pt was determined by ICP-OES. The %ID/g was calculated for comparison.

HPLC analysis of tumor tissues.
HeLa tumor-bearing mice were i.v. injected with P-CyPt (100 μM) or Pt IV NPs (100 μM) in 200 μL saline. Mice were euthanized and their tumors were resected after 4 h. The tumor tissues were cut into small pieces, to which NEM (100 mM, 200 μL), PBS buffer (200 μL) and RIPA buffer (200 μL) were added. The mixtures were sonicated in ice bath for 5 min to prepare the tumor homogenates. Then, 200 μL DMSO were added into the homogenates and sonicated for another 5 min to dissolve compounds. Finally, 200 uL methanol were added into the homogenates and centrifuged at 2,700× g for 10 min to remove proteins. The supernatant was injected into HPLC to analyze the compounds.
Fluorescence imaging of tumor tissue slices. Mice bearing s.c. HeLa tumors were i.v. injected with P-CyPt (100 μM) or Pt IV NPs (100 μM) in 200 μL saline, and the mice were sacrificed after 4 h. The tumors were resected and cut using a vibrating-blade microtome to obtain 10 μm-thickness slices. After staining with DAPI, the fluorescence images of tumor tissue slices were acquired with the Olympus IX73 fluorescent inverted microscope, using the microscope equipped with DAPI and Cy5.5 filters.
Histopathological staining. HeLa tumor-bearing mice were i.v. injected with PBS (200 μL), P-CyPt, Pt IV NPs, or cisplatin (2.25 mg kg -1 Pt). After 2 days, the HeLa tumors were excised and fixed in formalin. Then, the tumors were cryosectioned at 10-μm thickness and stained with anti-gammaH2A.X (ab81299, lot: GR3338944-19, dilution 1:200) at 4°C overnight. After being washed with PBS three times, the cells were then incubated with the Alexa Fluor®488-labeled secondary antibody (Alexa Fluor®488 goat anti-rabbit IgG (H+L), ab150077, lot: GR3376391-4, dilution 1:1000) at room temperature for 1 h. After that, Hoechst 33342 was used to stain the cell nuclei), H&E and TUNNEL (KeyGen Biotech. Co. Ltd., Nanjing, China) staining kit, respectively, according to the manufacturer's instructions. The confocal fluorescence using the IX73 optical microscope equipped with DAPI and FITC filters, microscope (Olympus, IX73) with U plan semi Apochromat phase objective 10x/0.3 dry lens (Olympus), and captured at 8 bit on a DP80 detector (Olympus), the analysis of the pictures captured using the microscope equipped with DAPI and FITC filters.
To examine the potential side effects, major organs including liver, spleen, kidney, heart, and lung were resected from, mice on 21-day post 5-dose treatment with PBS, P-CyPt, Pt IV NPs, or cisplatin (2.25 mg kg -1 Pt), and applied for histopathological analysis.
Blood analysis. Mice were i.v. injected with P-CyPt, Pt IV NPs, CDDP (2.25 mg kg -1 Pt) or PBS. The bloods (~1.0 mL) were collected from the venous sinus at 24 h postinjection, and preserved into 1.5-mL EDTA coated Eppendorf tubes that are chilled on ice. The collected blood samples were divided into two parts. Part one was applied for blood count analysis. Part two was centrifuged at 2,400 × g for 20 min. Biochemistry relative biomarkers including alanine aminotransferase (ALT), glutamic oxaloacetic transaminase (AST), γ-glutamyl transpeptidase (GGT), total bilirubin (TBil), albumin (Alb), total protein (TP), blood urea nitrogen (BUN) and creatinine (CRE) were determined using ELISA kits according to manufacturer's protocol.
Determination of the blood circulation half-life. P-CyPt, Pt IV NPs, or CDDP (at a dose of 2.25 mg kg -1 Pt) was i.v. injected into healthy female BALB/c nude mice (6-8 weeks old). After injection at 0, 0.5, 1, 2, 4, 8, 12, and 24 h, around 1.2 mL of bloods from every three mice at each time point were collected from the venous sinus and preserved into 1.5-mL EDTA-coated Eppendorf tubes that are chilled on ice. The collected blood was digested with concentrated HNO3 (65-70%, 2 mL) and HCl (36~38%, 1 mL) at 90 °C overnight. The residues were then diluted with 5 mL 2% HNO3 and 5% HCl solution, and the concentrations of Pt element were determined by ICP-OES, which were further applied to calculate the contents of Pt element in the blood. The plot of the percentage of Pt element remained in the blood versus injection time to yield the pharmacokinetic time.
Urine and Faeces analysis. Healthy female BALB/c nude mice were i.v. injected with P-CyPt, Pt IV NPs or CDDP (2.25 mg kg -1 Pt, dissolved in saline) and placed in metabolic cages for 12 h. Urine and faeces were collected at 2 (for 0-2 h), 4 (for 2-4 h), 8 (for 4-8 h) and 12 h (for 8-14 h), which were diluted in methanol and centrifuged at 3,600 × g for 10 min, followed by filtering via a 0.22 μm syringe filter. The solutions were analyzed by HPLC (660 nm detection) to characterize all the mCy-containing compounds. Using the established standard curves for P-CyPt, CyPt and Cy-COOH, the amount of each compound in the urine and faeces could be calculated, which were further applied to determine the percentage of them in the urine and faeces. The data were summarized in Supplementary Tables 5-8. Healthy female BALB/c nude mice were i.v. injected with P-CyPt, Pt Ⅳ NPs or CDDP (2.25 mg kg -1 Pt, dissolved in saline) and placed in metabolic cages for 12 h. Urine and faeces were collected and weighed. The urine and faeces were digested with concentrated HNO3 (65~70%) and HCl (36~38%) at 90 °C for 24 h. The solution was diluted to 5 mL 2% HNO3 and 5% HCl solution, and the concentration of Pt element was measured by ICP-OES, which was further applied to calculate the percentage of Pt in corresponding urine and faeces. The data were summarized in Supplementary Tables  9 and 10